Hemoregulatory compounds

ABSTRACT

The present invention relates to novel compounds which have hemoregulatory activities and can be used to stimulate hematopoiesis and for the treatment of viral, fungal and bacterial infectious diseases.

This application is a 371 of PCT/US96/18342, filed Nov. 12, 1996, whichclaims the benefit of priority from Provisional Application Ser. No.60/006,572, filed Nov. 13, 1995.

FIELD OF THE INVENTION

The present invention relates to novel compounds which havehemoregulatory activities and can be used to stimulate hematopoiesis andfor the treatment of viral, fungal and bacterial infectious diseases.

BACKGROUND OF THE INVENTION

The hematopoietic system is a life-long cell renewal process whereby adefined stem cell population gives rise to a larger population ofmature, differentiated blood cells (Dexter T M. Stem cells in normalgrowth and disease. Br Med J 1987; 195:1192-1194) of at least ninedifferent cell lineages (erythrocytes, platelets, eosinophils,basophils, neutrophils, monocytes/macrophages, osteoclasts, andlymphocytes) (Metcalf D. The Molecular Control of Blood Cells. 1988;Harvard University Press, Cambridge, Mass.). Stem cells are alsoultimately responsible for regenerating bone marrow following treatmentwith cytotoxic agents or following bone marrow transplantation.

The major dose-limiting toxicities of most standard anti-neoplasticdrugs are related to bone marrow suppression, which if severe andprolonged, can give rise to life-threatening infectious and hemorrhagiccomplications. Myelosuppression is predictable and has been reported tobe dose-limiting in greater than 50% of single-agent Phase I trialscytotoxic compounds (Merrouche Y, Catimel G, Clavel M. Hematopoieticgrowth factors and chemoprotectants; should we move toward a two-stepprocess for phase I clinical trials in oncology? Ann Oncol 1993;4:471-474). The risk of infection is directly related to the degree ofmyelosuppression as measured by the severity and duration of neutropenia(Brody G P, Buckley M, Sathe Y S, Freireich E J. Quantitativerelationship between circulating leukocytes and infections with acuteleukemia. Ann In Med 1965; 64:328-334).

The control of hematopoiesis involves the interplay of a variety ofcytokines and growth factors during various stages of the hematopoieticcascade, including early pluripotent stem cells and mature circulatingeffector cells. These regulatory molecules include granulocyte colonystimulating factor (G-CSF), granulocyte-macrophage stimulating factor(GM-CSF), macrophage-colony stimulating factor (M-CSF), and a variety ofinterleukins which have overlapping, additive and synergistic actionswhich play major roles in host defence. Mechanistically, this isaccomplished by enhancing the production of granulocytes andmacrophages, as well as by the activation of effector cell functions(Moore M A S. Hemopoietic growth factor interactions: in vitro and invivo preclinical evaluation. Cancer Surveys 1990; 9:7-80). Thesecoordinated activities support optimal host defences which are necessaryfor fighting bacterial, viral and fungal infections.

Strategies to prevent or reduce the severity of neutropenia andmyelotoxicity include the use of hematopoietic growth factors and/orother hematopoietic cytokines. Such treatments are becoming commonpractice, in that they offer the potential of increased doses ofcytotoxic agents that may improve the therapeutic efficacy ofantineoplastic agents, and reduce the morbidity associated with theiruse (Steward W P. Granulocyte and granulocyte-macrophage colonystimulating factors, Lancet 1993; 342:153-157). Clinical studies havedemonstrated the G-, GM- and/or M-CSF may reduce the duration ofneutropenia, accelerate myeloid recovery, and reduceneutropenia-associated infections and other infectious complications inpatients with malignancies who are receiving cytotoxic chemotherapy orin high infectious-risk patients following bone marrow transplantation(Steward W P. Granulocyte and granulocyte-macrophage colony stimulatingfactors, Lancet 1993; 342:153-157 and Munn D H, Cheung N K V.Preclinical and clinical studies of macrophage colong-stimulatingfactor. Semin Oncol 1992; 19:395-407).

Synthetic peptides have been reported to induce the synthesis andrelease of haematoporetic mediators, including m-CSF from bone marrowstromal elements see U.S. patent application Ser. No. 08/001,905.

We have now found certain novel non-peptide compounds which have astimulative effect on myelopoietic cells. They are useful in stimulatingmyelopoiesis in patients suffering from reduced myelopoietic activity,including bone marrow damage, agranulocytosis and aplastic anemiaincluding patients having depressed bone maprow function due toimmunosuppressive treatment to suppress tissue reactions i.e. in bonemarrow transplant surgery. They may also be used to promote more rapidregeneration of bone marrow after cytostatic chemotherapy and radiationtherapy for neoplastic an d viral diseases. They may be of particularvalue where patients have serious infections due to a lack of immuneresponse following b one marrow failure . They are useful in thetreatment and pievention of viral, fungal and bacterial disease.

SUMMARY OF THE INVENTION

This invention comprises compounds, hereinafter represented as Formula(I), which have hemoregulatory activities and can be used to stimulatehematopoiesis and in the prevention and treatment of bacterial, viraland fungal diseases.

These compounds are useful in the restoration of leukocytes in patientswith lowered cell counts resulting from a variety of clinicalsituations, such as surgical induced myclosuppression, AIDS, ARDS,congenital myelodysplacis, bone marrow and organ transplants; in theprotection of patients with leukopenia from infection; in the treatmentof severely burned patients and in the amelioration of themyelosuppression observed with some cell-cycle specific antiviral agentsand in the treatment of infections in patients who have had bone marrowtrasplants, especially those with graft versus host disease, in thetreatment of tuberculosis and in the treatment of fevers of unknownorigin in humans and animals. The compounds are also useful in thetreatment and prevention of viral, fungal and bacterial infectiousdiseases, particularly Candida and Herpes in both immunosuppressed and“normal” subjects. They are useful in the treatment of sepsis caused bygram negative and gram positive organisms.

These compounds may also be used in combination with the myelosuppresiveagents of co-pending U.S. application Ser. No. 07/99,465 and U.S. Pat.No. 4,499,081, incorporated by reference herein, to provide alternatingpeaks of high and low activity in bone marrow cells, thus augmenting thenatural circadian rhythm of hematopoiesis. In this way, cytostatictherapy can be given at periods of low bone marrow activity, thusreducing the risk of bone marrow damage, while regeneration will bepromoted by the succeeding peak of activity. This invention is also apharmaceutical composition, which comprises a compound of Formula (I)and a pharmaceutically acceptable carrier.

This invention further constitutes a method for stimulating themyelopoietic system of an animal, including humans, which comprisesadministering to an animal in need thereof, an effective amount of acompound of Formula (I).

This invention also constitutes a method for preventing and treatingviral, fungal and bacterial infections in immunosuppressed and normalanimals, including humans, which comprises administering to an animal inneed thereof, an effective amount of a compound of Formula (I).

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention are represented by structural Formula I

A¹ and A² are independently Z—(CH₂)_(k)—(NR²)_(y)—.

Z is independently a 4-10 membered mono- or bicyclic heterocyclic ringsystem containing up to four heteroatoms N, O, S in the ring in which atleast one heteroatom is N, and wherein the ring is substituted orunsubstituted by one or two C₁₋₄alkyl, F, Cl, Br, I, C₁₋₄ alkoxy,(CH₂)_(m)R₄, oxo, oxime, O—C₁₋₄alkyloxime, hydroxy, N(R₃)₂, acylamino oraminoacyl groups, 8, 9, 10 membered monocyclic ring systems beingexcluded;

R¹ and R² are independently hydrogen, C₁₋₄alkylC(O)R₄, C₁₋₄alkyl or R₁and R₂ are benzyl which is optionally substituted by one or twoC₁₋₄alkyl, C₁₋₄alkoxy, F, Cl, I, Br, OH, or N(R₃)₂;

R₃ is independently hydrogen, C₁₋₄alkyl, or benzyl;

R₄ is independently OR₃, N(R₃)₂ or SR₃; and

k is an integer from 0 to 4;

m is independently an integer from 1 to 3;

n is 1 or 2;

y is zero or one;

or a pharmaceutically acceptable salt thereof.

C₁₋₄ alkyl groups may be straight or branched.

The compounds of the present inventiion may contain one or moreasymmetric carbon atoms and may exist in racemic and optically activeform. All these compounds and diastereomers are contemplated to bewithin the scope of the present invention.

Z in the above Formula (I) denotes an optionally substituted pyrrolyl,isopyrrolyl, pyrazolyl, isoimidazolyl, triazolyl, isoxazolyl, oxazolyl,thiazolyl, isothiazolyl, oxadiazolyl, pyridinyl, pyridazinyl,pyrimidinyl, pyrazinyl, pyrrolidinyl, piperazinyl, triazinyl,morpholinyl, indolyl, indoleninyl, isobenzazolyl, pyrindinyl,ioindazolyl, indoxazinyl, benzoxazolyl, quinolinyl, isoquinolinyl,cinnolinyl, quinazolinyl, naphthyridinyl, pyridopyridinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, indolinyl,2-pyrrolidonyl, imidazolyl, imidazolidinyl, imidazolinyl, piperidyl,tetrazolyl, quinuclidinyl, azetidinyl, or purinyl;

Preferred compounds are those wherein Z is optionally substitutedpyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolinyl,tetrahydroquinolinyl, azetidinyl, or pyrrolidinyl;

More preferred compounds are those wherein Z is optionally substituted2-pyridinyl, 2-pynmidinyl, ²-pyrazinyl, 2-pyrrolidon-5-yl, orpyrrolidinyl.

Preferred compounds are N,N′-bis(picolinoyl)-1,4-diazacycloheptane,N,N′-bis(picolinoyl)piperazine, N,N′-bis(pyroglutamoyl)piperazine orN,N′-bis(pyroglutamoyl)-1,4-diazacycloheptane.

Methods of Preparation

Compounds of Formula (I) wherein A¹, A² and m are defined as in Formula(I) are prepared by methods analogous to those described in Scheme 3.Appropriate diamines (such as 1 in Scheme 3) are bis-acylated with asuitable heterocyclic acid (such as 2 in Scheme 3) using an activatingagent (such as BOP reagent) with a suitable base (such as iPr₂NEt) in apolar aprotic solvent (such as DMF) to give final product.

In order to use a compound of the Formula (I) or a pharmaceuticallyacceptable salt thereof for the treatment of humans and other mammals itis normally formulated in accordance with standard pharmaceuticalpractice as a pharmaceutical composition.

According to a still further feature of the present invention there areprovided pharmaceutical compositions comprising as active ingredient oneor more compounds of Formula (I) as herein before defined orphysiologically compatible salts thereof, in association with apharmaceutical carrier or excipient. The compositions according to theinvention may be presented for example, in a form suitable for oral,nasal, parenteral or rectal administration.

As used herein, the term “pharmaceutical” includes veterinaryapplications of the invention. These compounds may be encapsulated,tableted or prepared in an emulsion or syrup for oral administration.Pharmaceutically acceptable solid or liquid carriers may be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition. Liquid carriers include syrup, peanut oil, olive oil,glycerin, saline and water. Solid carriers include starch, lactose,calcium sulfate dihydrate, terra alba, magnesium stearate or stearicacid, talc, pectin, acacia, agar or gelatin. The carrier may alsoinclude a sustained release material such a glyceryl monostearate orglyceryl distearate, alone or with a wax. The amount of solid carriervaries but, preferably will be between about 20 mg to about 1 g perdosage unit. The pharmaceutical preparations are made following theconventional techniques of pharmacy involving milling, mixing,granulating, and compressing, when necessary, for tablet forms; ormilling, mixing and filling for hard gelatin capsule forms. Capsulescontaining one or several active ingredients may be produced, forexample, by mixing the active ingredients with inert carriers, such aslactose or sorbitol, and filling the mixture into gelatin capsules. Whena liquid carrier is used, the preparation will be in the form of asyrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such aliquid formulation may be administered directly p.o. or filled into asoft gelatin capsule. Organ specific carrier systems may also be used.

Alternately pharmaceutical compositions of the compounds of thisinvention, or derivatives thereof, may be formulated as solutions oflyophilized powders for parenteral administration. Powders may bereconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. The liquid formulationis generally a buffered, isotonic, aqueous solution. Examples ofsuitable diluents are normal isotonic saline solution, standard 5%dextrose in water or buffered sodium or ammonium acetate solution. Suchformulation is especially suitable for parenteral administration, butmay also be used for oral administration and contained in a metered doseinhaler or nebulizer for insufflation. It may be desirable to addexcipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose,acacia, polyethylene glycol, mannitol, sodium chloride or sodiumcitrate.

For rectal administration, a pulverized powder of the compounds of thisinvention may be combined with excipients such as cocoa butter,glycerin, gelatin or polyethylene glycols and molded into a suppository.The pulverized powders may also be compounded with an oily preparation,gel, cream or emulsion, buffered or unbuffered, and administered througha transdermal patch.

Nasal sprays may be formulated similarly in aqueous solution and packedinto spray containers either with an aerosol propellant or provided withmeans for manual compression.

Dosage units containing the compounds of this invention preferablycontain 0.05-50 mg, for example 0.05-5 mg of the compound of formula (V)or salt thereof.

According to a still further feature of the present invention there isprovided a method of stimulation of myelopoiesis which comprisesadministering an effective amount of a pharmaceutical composition ashereinbefore defined to a subject.

No unacceptable toxicological effects are expected when compounds of theinvention are administered in accordance with the present invention.

The biological activity of the compounds of Formula (I) are demonstratedby the following tests.

Induction of Hematopoietic Synergistic Activity in Stromal Cells

The murine bone marrow derived stromal cell line, C6.4 is grown in 12well plates in RPMI 1640 with 10% FBS. Upon reaching confluence, theC6.4 cells are washed and the media exchanged with fresh RPMI 1640without FBS. Confluent cell layers of murine C6.4 cells are treated withcompound. Cell-free supernatants are collected 18 hours later.Supernatants are fractionated with a Centricon-30 molecular weightcut-off membrane. C6.4 cell hematopoietic synergistic factor (HSF)activity is measured in a murine CFU-C assay.

CFU-C Assay

Bone marrow cells are obtained from C57B 1/6 female mice and suspendedin RPMI 1640 with 10% FBS. Bone marrow cells (7.5E+4 cells/mL) arecultured with sub optimal levels of CFU plus dilutions of test C6.4 cell30K-E supernatants from above in a standard murine soft agar CFU-Cassay. Cell aggregates >50 cells are counted as colonies. The number ofagar colonies counted is proportional to the amount of HSF presentwithin the C6.4 bone marrow stromal line supernatant.

Effector Cell Function Assay

Female C57B1 mice are administered test compound IP or PO daily for 8days. Resident peritoneal exudate cells (PEC) utilized ex vivo fromtreated or untreated mice are harvested with cold calcium andmagnesium-free DPBS supplemented with heparin and antibiotics within 2-4hours following the last injection. Adherent PEM populations areprepared by incubating standardized PEC suspensions in microtiter dishesfor 2 hours at 37° C. (5% CO₂) and removing nonadherent cells by washingthe wells with warm buffer.

The superoxide dismutase-inhibitable (SOD) superoxide released byeffector cells in response to a in vitro stimulation by phorbolmyristate acetate (PMA) (100-200 nM) or pre-opsonized (autologous sera)live C. albicans (E:T=1:10) are quantitated in a microtiterferricytochrome c reduction assay. The assay is performed in thepresence of 1% gelatin/HBSS and 80 uM ferricytochrome c in a totalvolume of 200 uL/well. The nmoles of cytochrome c reduced/well iscalculated from spectrophotometric readings (550 nm) taken following a 1hour incubation at 37° C. (5% CO₂). The amount of SOD-inhibitablecytochrome c reduced is determined by the inclusion of wells containingSOD (200 U/well). Baseline superoxide release is determined in theabsence of stimuli. Experimental data are expressed as a percentage ofthe control group.

The following examples are illustrative and are not limiting of thecompounds of this invention.

EXAMPLE 1 N,N′-Bis(picolinoyl)-1,4-diazacycloheptane

To a solution of 1,4-diazacycloheptane (0.31 g, 3.08 mmol) in pyridine(30 mL) was added picolinic acid (0.74 g, 6.01 mmol) and EDC (1.16 g,6.05 mmol). After 18 h at room temperature, the bulk of the pyridine wasremoved in vacuo. The residue was dissolved in water (50 mL) andextracted with EtOAc (3×50 mL). The combined organic extracts werewashed with water (50 mL) and dried over Na₂SO₄. Removal of solvent gave0.14 g of a red syrup. Purification by flash chromatography (20 %MeOH/EtOAc, silica gel) afforded 0.09 g (10%) of the title compound. MS(ES+) m/z 311.2 [M+H]⁺.

EXAMPLE 2 N,N′-Bis(picolinoyl)piperazine

In a manner analogous to Example 1, piperazine (0.09 g, 1.04 mmol),picolinic acid (0.37 g, 3.00 mmol) and EDC (0.57 g, 3.00 mmol) inpyridine (10 mL) gave 0.29 g (94 %) of the title compound. MS (ES+) m/z297.0 [M+H]⁺.

EXAMPLE 3

Formulations for pharmaceutical use incorporating compounds of thepresent invention can be prepared in various forms and with numerousexcipients. Examples of such formulations are given below.

Tablets/Ingredients Per Tablet 1. Active ingredient 0.5 mg (Cpd of Form.I) 2. Corn Starch  20 mg 3. Alginic acid  20 mg 4. Sodium alginate  20mg 5. Mg stearate 1.3 mg

Procedure for tablets:

Step 1 Blend ingredients No. 1, No. 2, No. 3 and No. 4 in a suitablemixer/blender.

Step 2 Add sufficient water portion-wise to the blend from Step 1 withcareful mixing after each addition. Such additions of water and mixinguntil the mass is of a consistency to permit its converion to wetgranules.

Step 3 The wet mass is converted to granules by passing it through anoscillating granulator using a No. 8 mesh (2.38 mm) screen.

Step 4 The wet granules are then dried in an oven at 140° F. (60° C.)until dry.

Step 5 The dry granules are lubricated with ingredient No. 5.

Step 6 The lubricated granules are compressed on a suitable tabletpress.

Parenteral Formulation

A pharmaceutical composition for parenteral administration is preparedby dissolving an appropriate amount of a compound of Formula I inpolyethylene glycol with heating. This solution is then diluted withwater for injections Ph Eur. (to 100 ml). The solution is thensterilized by filtration through a 0.22 micron membrane filter andsealed in sterile containers.

What is claimed is:
 1. A method of treating viral, fungal or bacterialinfections which comprises administering to an animal in need thereof,an effective amount of a compound of Formula (I):

wherein: A¹ and A² are independently Z—(CH₂)_(k)—(NR₂)_(y)—; Z isindependently 2-pyridinyl, 2-pyrimidinyl, 2-pyrazinyl 2-pyrrolidon-5-yl,or 2-pyrrolidinyl, wherein the ring is substituted or unsubstituted byone or two C₁₋₄alkyl, F, Cl, Br, I, C₁₋₄ alkoxy, (CH₂)_(n)R₄, oxo,oxime, O—C₁₋₄alkyloxime, hydroxy, N(R₃)₂, acylamino or aminoacyl groups;R² is hydrogen, C₁₋₄alkylC(O)R₄, C₁₋₄alkyl or R² is benzyl which isoptionally substituted by one or two C₁₋₄alkyl, C₁₋₄alkoxy, F, Cl, I,Br, OH, or N(R₃)₂; R₃ is independently hydrogen, C₁₋₄alkyl, or benzyl;R₄ is independently OR₃, N(R₃)₂ or SR₃; and k, is an integer from 0 to4; m is 2; n is independently 2 or 3; y is zero or one; or apharmaceutically acceptable salt thereof.
 2. A method of treatmentaccording to claim 1 wherein the compound is selected fromN,N′-bis(picolinoyl)piperazine, or N,N′-bis(pyroglutamoyl)piperazine. 3.A method of treating sepsis which comprises administering to an animalin need thereof, an effective amount of a compound of Formula (I):

wherein: A¹ and A² are independently Z—(CH₂)_(k)—(NR²)_(y)—; Z isindependently 2-pyridinyl, 2-pyrimidinyl, 2-pyrazinyl,2-pyrrolidon-5-yl, or 2-pyrrolidinyl, wherein the ring is substituted orunsubstituted by one or two C₁₋₄; alkyl, F, Cl, Br, I, C₁₋₄ alkoxy,(CH₂)_(n)R₄, oxo, oxime, O—C₁₋₄alkyloxime, hydroxy, N(R₃)₂, acylamino oraminoacyl groups; R² is hydrogen, C₁₋₄alkylC(O)R₄, C₁₋₄alkyl or R² isbenzyl which is optionally substituted by one or two C₁₋₄alkyl ,C₁₋₄alkoxy, F, Cl, I, Br, O or N(R₃)₂; R₃ is independently hydrogen,C₁₋₄alkyl, or benzyl; R₄ is independently OR₃, N(R₃)₂ or SR₃; and k isan integer from 0 to 4; m is 2; n is independently 2 or 3; y is zero orone; or a pharmaceuticadly acceptable salt thereof.
 4. A method ofstimulating myclopoiesis which comprises administering to an animal inneed thereof, an effective amount of a compound of Formula (I):

wherein: A¹ and A² are independently Z—(CH₂)_(k)—(NR²)_(y)—; Z isindependently 2-pyridinyl, 2-pyrimidinyl, 2-pyrazinyl,2-pyrrolidon-5-yl, or 2-pyrrolidinyl, wherein the ring is substituted orunsubstituted by one or two C₁₋₄alkyl, F, Cl, Br, I, C₁₋₄ alkoxy,(CH₂)_(n)R₄, oxo, oxime, O—C₁₋₄alkyloxime, hydroxy, N(R₃)₂, acylamino oraminoacyl groups; R² is hydrogen, C₁₋₄alkylC(O)R₄, C₁₋₄alkyl or R² isbenzyl which is optionally substituted by one or two C₁₋₄alkyl,C₁₋₄alkoxy, F, Cl, I, Br, OH, or N(R₃)₂; R₃ is independently hydrogen,C₁₋₄alkyl, or benzyl, R₄ is independently OR₃, N(R₃)₂ or SR₃, and k isan integer from 0 to 4; m is 2; n is independently 2 or 3; y is zero orone; or a pharmaceutically acceptable salt thereof.